On-line solid phase extraction/capillary reverse-phase liquid chromatography has been used as a very important technological system in studying proteomes by virtue of its high efficiency in separation. Particularly, this allows efficient separation of trace amounts of biological substances and enables highly efficient identification of trace amounts of proteins due to its broad spectrum of analyte-solid phase interactions.
As a method for analyzing proteins, a mass spectrometry-based method has served as a standard analytic platform of proteomic study. A typical example of the method, such as a shot-gun method or a bottom-up method, includes hydrolysis of proteins into peptides prior to the analysis by a mass spectrometer. Such hydrolysis increases the solubilities of biological samples and produces peptide fragments that may be ionized and detected easily in a mass spectrometer.
However, the aforementioned method inevitably increases the complexity of samples. For example, in the case of one of the simplest proteomes, yeast proteome, 300,000 or more peptide fragments are produced from about 6,000 expressed proteins. Therefore, in order to overcome undersampling issue in analyzing the samples with enormous complexities, various methods including on-/off-line multidimensional protein identification technology have been developed (Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., et al., Nat. Biotechnol. 1999, 17, 676-682; Chen, E. I., Hewel, J., Felding-Habermann, B., Yates, J. R. III, Mol. Cell. Proteomics 2006, 5, 53-56). However, improvement in the efficiency and sensitivity of a liquid chromatography column is still required. In this context, it has been known that the sensitivity of analysis based on liquid chromatography/mass spectrometry may be increased rapidly when the inner diameter of a separation column is decreased while maintaining the length thereof (Kim, M.-S., Choie, W.-S., Shin, Y. S., Yu, M. H., Lee, S.-W., Bull. Korean Chem. Soc. 2004, 25, 1833-1839).
In addition, in the case of a biological sample containing a significant amount of detergents and salts, an on-line desalting operation is an essential process required prior to mass spectrometry. This is because such impurities interrupt ionization of the peptide sample to be analyzed by a mass spectrometer, resulting in a drop in mass spectrometric detection sensitivity for the peptide sample. Thus, considering time saving and sample loss, on-line desalting is more suitable than off-line desalting.
Meanwhile, reverse-phase liquid chromatography systems according to the related art merely have a one-dimensional separation function of desalting and concentrating a sample by using a solid-phase extraction column. Even in the case of a two-dimensional reverse-phase liquid chromatography system carrying out a two-dimensional separation function by using a strong cation exchange chromatography system on-line linked to a reverse-phase liquid chromatography system, it is difficult to perform precise analysis due to mutual interference. Otherwise, such systems essentially require multiple solvent feed pumps, and thus have a complicated structure due to multiple valves for controlling the pumps. Moreover, such two-dimensional on-line reverse-phase liquid chromatography systems that also have Ion-line digestion function and online enrichment of phosphopeptides have never been reported or demonstrated before.